Method for preparing polymer maleimides

ABSTRACT

Methods for preparing polymeric reagents bearing a maleimide are provided. Also provided are compositions comprising the polymeric reagents, and conjugates prepared by polymeric reagents obtained by the described methods.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of priority to U.S. provisionalpatent application Ser. No. 60/700,972, the contents of which areincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to methods for preparing water soluble andnon-peptidic polymers carrying maleimide functional groups, particularlymaleimide-terminated poly(ethylene glycol) polymers, and to compositionsand formulation containing the same.

BACKGROUND OF THE INVENTION

Maleimides are versatile derivatives that find extensive use in chemicalsynthesis and in biological and pharmacological applications. As Michaelacceptors, maleimides react readily with sulfhydryl groups to formstable thioether bonds. This reaction is extensively used with proteinsand the like where both sulfhydryl and amine groups are present. Atapproximately neutral pH, maleimides are highly selective, withsulfhydryl groups being about 1,000 times more reactive than aminegroups (Smyth et al., Biochem. J., 91, 589, 1964; Gorin et al. Arch.Biochem. Biophys. 115, 593, 1966; Partis et al., J. Protein Chem, 2,263-277, 1983). At higher pH values of 8 or above, the reaction ofmaleimides with amine groups begins to significantly compete (Brewer andRiehm, Anal. Biochem. 18, 248, 1967). While best known as Michaelacceptors, maleimides are also useful for their reactivity asdienophiles (Baldwin et al., Tetrahedron Lett., 32, 5877, 1991; Philpand Robertson, J. Chem. Soc., Chem. Commun., 1998, 879; Bravo et al.,Heterocycles, 53, 81, 2000) and as dipolarophiles (Grigg et al., J.Chem. Soc., Perkin Trans. 1, 1988, 2693; Konopikova et al., Collect.Czech. Chem. Commun., 57, 1521, 1991; Philp and Booth, TetrahedronLett., 39, 6987, 1998).

Maleimide groups can be used to facilitate covalent attachment ofproteins and other molecules to polymers. For example, the hydrophilicpolymer “poly(ethylene glycol)”, abbreviated as “PEG”, is often used toconjugate bioactive molecules and render them soluble in aqueous media(Harris et al. “Poly(Ethylene Glycol) Chemistry and BiologicalApplications”, ACS Symposium Series, ACS, Washington, D.C., 1997).PEG-maleimide is an example of a reactive polymer suitable for reactionwith thiol or amino groups on a biologically active molecule.

Many of the methods for preparing PEG maleimides involve connecting anactivated PEG to a small linker molecule comprising a maleimide group,many of which are available commercially. There are a variety ofshortcomings associated with several known PEG maleimides and methodsfor their production. For example, the so-called “linkerless” PEGmaleimides, which have no linker group between the PEG and the maleimidegroup, are often prepared directly from a PEG amine using one of twomethods. See U.S. Pat. No. 6,602,498. These methods, however, generallyresult in a relatively impure product inasmuch as a fairly significantamount of an open ring maleamic acid-containing derivative is present inthe final product as will be discussed below.

In the first method disclosed in U.S. Pat. No. 6,602,498, a watersoluble and non-peptidic polymer backbone is reacted with maleicanhydride to form an open ring amide carboxylic acid intermediate (amaleamic acid intermediate). The ring of the intermediate is then closedin a second step by heating the intermediate in the presence of aceticanhydride and a salt of acetic acid, such as sodium or potassiumacetate, to a temperature of about 50° C. to about 140° C. for about 0.2to about 5 hours. This two-step process is summarized in the ReactionScheme I, provided below:

The crude maleimide-terminated, water-soluble polymer-containingcomposition made by this method may contain a substantial amount of theopen ring maleamic acid intermediate. A major cause for the appearanceof the open ring maleamic acid intermediate may lie with the heatingstep, especially if any acidic species is generated or is a contaminantin the acetic anhydride. Under these conditions, it is possible toisomerize the C═C bond and thus make ring closure difficult, if notimpossible. As a result, it is desirable to purify the polymer productby some method, such as ion exchange chromatography, capable of removingthe impurity. However, the maleimide ring system does not tolerate achromatographic column bearing basic or nucleophilic sites, thus makingpurification more difficult. A second problem with this synthetic routestems from the use of PEG amine.

Similarly, Sakanoue et al., U.S. Patent Application Publication No.2003/0065134 A1, describes a related method except that thePEG-maleimides produced therein comprise a propylene group rather thanan ethylene group between the ultimate PEG oxygen and the maleimidenitrogen. The method described in Sakanoue et al., however, suffers fromthe same problems as mentioned above. Further, the reference teachesthat the PEG amines are generally manufactured by reduction of a nitrilegroup using hydrogen and a nickel catalyst, which can lead to theintroduction of additional impurities due to reaction between the amineproduct and an imine intermediate.

In a second synthetic route described in U.S. Pat. No. 6,602,498 (the“Aqueous N-alkoxycarbonylmaleimide route”), an N-alkoxycarbonylmaleimideis reacted with a polymeric amine to form a maleimide-terminated,water-soluble polymer product. A ring-opening and ring-closing reactionoccurs similar to the one described above. The reaction is conductedover a slowly increasing temperature gradient in an aqueous sodiumbicarbonate buffer at a pH of about 8.5. The maleimide group, however,is not stable at those conditions and undergoes hydrolysis to maleamicacid. Therefore, two parallel reactions occur during synthesis:formation of maleimide ring and maleimide ring hydrolysis.

One approach for addressing the problem would be to stop the reaction ata time when a maximum amount of the maleimide-terminated, water-solublepolymer product is formed. While this approach appears sound in theory,it is almost an impossible task in commercial practice due to thechanging reaction temperature wherein it can be difficult toreproducibly achieve the temperature gradient during consecutivemanufacturing batches. For example, consecutive commercial batches ofcertain maleimide-terminated, water-soluble polymers were found to havemaleimide purity from 65 to 80%, and maleamic acid content of about 20to 35%. Again, chromatography is not a viable option because of thesensitivity of the maleimide group to the functional groups of the ionexchange column. Furthermore, even if it were possible to reproduciblycontrol the temperature gradient and stop the reaction at the propertime, the approach requires close monitoring and additional equipment(e.g., thermocouples, heat jackets, and so forth), thereby addingcomplexity to the approach.

Other approaches for preparing maleimide-terminated, water-solublepolymers are described in International Patent Publication WO 05/056636.In one approach (labeled as “Reaction Scheme II” below) a polymercomprising a leaving group (“LG”) and a salt of an imide (shown as thepotassium salt of a tricyclic amide) are reacted via nucleophilicsubstitution to form a polymer intermediate, which is then followed by areverse Diels-Alder reaction to provide a maleimide functionalizedpolymer and furan.

Although the reaction shown above utilizes relatively simplefunctionalized polymers and Diels-Alder adduct reagents that react toform so-called “linkerless” maleimides (meaning the maleimide group isdirectly attached to the polymer), the reaction requires notcommercially available reagents.

Notwithstanding the approaches described above, there remains a need toprovide still other approaches for preparing maleimide-terminated,water-soluble polymers so that, for example, the approach best suitedfor a particular need can be used. The novel approach described hereinis believed to provide, among other things, maleimide-terminatedpolymers in high yield and free from significant amounts of polymericimpurities, particularly significant amounts of polymer impurities thatcannot be readily removed using conventional purification techniques,such as ion exchange chromatography.

SUMMARY OF THE INVENTION

In one or more embodiments, a method for preparing a substitutedmaleamic acid-terminated, water-soluble polymer is provided, the methodcomprising:

-   a) combining an amine-terminated, water-soluble polymer with a    maleimide reagent under substantially nonaqueous conditions to form    a substituted maleamic acid-terminated, water-soluble polymer; and-   a′) optionally, isolating the substituted maleamic acid-terminated,    water-soluble polymer.

In one or more embodiments, a method for preparing amaleimide-terminated, water-soluble polymer is provided, the methodcomprising:

-   a) combining an amine-terminated, water-soluble polymer with a    maleimide reagent under substantially nonaqueous conditions to form    a substituted maleamic acid-terminated, water-soluble polymer; and-   b) exposing the maleamic acid-terminated, water-soluble polymer to    elimination conditions under substantially nonaqueous conditions to    thereby result in a maleimide-terminated, water-soluble polymer.

In one or more embodiments of the invention, a maleimide-terminated,water-soluble polymer-containing composition is provided, thecomposition resulting from the method comprising:

-   a) combining an amine-terminated, water-soluble polymer with a    maleimide reagent under substantially nonaqueous conditions to form    a maleamic acid-terminated, water-soluble polymer; and-   b) exposing the maleamic acid-terminated, water-soluble polymer to    elimination conditions under substantially nonaqueous conditions to    thereby result in a maleimide-terminated, water-soluble    polymer-containing composition.

In one or more embodiments of the invention, a method for preparing aconjugate-containing composition is provided, the method comprisingcombining a thiol-containing biologically active agent with amaleimide-terminated, water-soluble polymer as provided herein tothereby result in a conjugate-containing composition.

In one or more embodiments of the invention, a conjugate-containingcomposition is provided, the composition resulting from the methodcomprising combining a thiol-containing active agent with amaleimide-terminated, water-soluble polymer-containing composition asprovided herein.

In one or more embodiments of the invention, a compound is provided, thecompound, in isolated form, has the following structure:

wherein:

POLY is a water-soluble polymer;

(b) is zero or one;

X¹, wherein present, is a spacer moiety;

Y¹ is O or S;

Y² is O or S;

(a) is an integer from 1 to 20;

R¹, in each instance, is independently H or an organic radical;

R², in each instance, is independently H or an organic radical;

R³, in each instance, is independently H or an organic radical; and

R⁴, in each instance, is independently H or an organic radical.

DETAILED DESCRIPTION OF THE INVENTION

Before describing one or more embodiments of the present invention indetail, it is to be understood that this invention is not limited to theparticular polymers, reagents, and the like, as such may vary.

It must be noted that, as used in this specification and the claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “apolymer” includes a single polymer as well as two or more of the same ordifferent polymers, reference to “a drying agent” refers to a singledrying agent as well as two or more of the same or different dryingagents, and the like.

In describing and claiming the present invention(s), the followingterminology will be used in accordance with the definitions providedbelow.

“PEG,” “polyethylene glycol” and “poly(ethylene glycol)” as used herein,are interchangeable. Typically, PEGs for use in accordance with theinvention comprise the following structure: “—(OCH₂CH₂)_(n)—” where (n)is 2 to 4000. As used herein, PEG also includes“—CH₂CH₂—O(CH₂CH₂O)_(n)—CH₂CH₂—” and “—(OCH₂CH₂)_(n)O—,” depending uponwhether or not the terminal oxygens have been displaced. Throughout thespecification and claims, it should be remembered that the term “PEG”includes structures having various terminal or “end capping” groups. Theterm “PEG” also means a polymer that contains a majority, that is tosay, greater than 50%, of —OCH₂CH₂— or —CH₂CH₂O— repeating subunits.With respect to specific forms, the PEG can take any number of a varietyof molecular weights, as well as structures or geometries such as“branched,” “linear,” “forked,” “multifunctional,” and the like, to bedescribed in greater detail below.

The terms “end-capped” and “terminally capped” are interchangeably usedherein to refer to a terminal or endpoint of a polymer having anend-capping moiety. Typically, although not necessarily, the end-cappingmoiety comprises a hydroxy or C₁₋₂₀ alkoxy group, more preferably aC₁₋₁₀ alkoxy group, and still more preferably a C₁₋₅ alkoxy group. Thus,examples of end-capping moieties include alkoxy (e.g., methoxy, ethoxyand benzyloxy), as well as aryl, heteroaryl, cyclo, heterocyclo, and thelike. It must be remembered that the end-capping moiety may include oneor more atoms of the terminal monomer in the polymer [e.g., theend-capping moiety “methoxy” in CH₃(OCH₂CH₂)_(n)—]. In addition,saturated, unsaturated, substituted and unsubstituted forms of each ofthe foregoing are envisioned. Moreover, the end-capping group can alsobe a silane. The end-capping group can also advantageously comprise adetectable label. When the polymer has an end-capping group comprising adetectable label, the amount or location of the polymer and/or themoiety (e.g., active agent) to which the polymer is coupled can bedetermined by using a suitable detector. Such labels include, withoutlimitation, fluorescers, chemiluminescers, moieties used in enzymelabeling, colorimetric moieties (e.g., dyes), metal ions, radioactivemoieties, and the like. Suitable detectors include photometers, films,spectrometers, and the like. The end-capping group can alsoadvantageously comprise a phospholipid. When the polymer has anend-capping group comprising a phospholipid, unique properties areimparted to the polymer and the resulting conjugate. Exemplaryphospholipids include, without limitation, those selected from the classof phospholipids called phosphatidylcholines. Specific phospholipidsinclude, without limitation, those selected from the group consisting ofdilauroylphosphatidylcholine, dioleylphosphatidylcholine,dipalmitoylphosphatidylcholine, disteroylphosphatidylcholine,behenoylphosphatidylcholine, arachidoylphosphatidylcholine, andlecithin.

“Non-naturally occurring” with respect to a polymer as described herein,means a polymer that in its entirety is not found in nature. Anon-naturally occurring polymer may, however, contain one or moremonomers or segments of monomers that are naturally occurring, so longas the overall polymer structure is not found in nature.

The term “water soluble” as in a “water-soluble polymer” is any polymerthat is soluble in water at room temperature. Typically, a water-solublepolymer will transmit at least about 75%, more preferably at least about95%, of light transmitted by the same solution after filtering. On aweight basis, a water-soluble polymer will preferably be at least about35% (by weight) soluble in water, more preferably at least about 50% (byweight) soluble in water, still more preferably about 70% (by weight)soluble in water, and still more preferably about 85% (by weight)soluble in water. It is most preferred, however, that the water-solublepolymer is about 95% (by weight) soluble in water or completely solublein water.

Molecular weight in the context of a water-soluble polymer, such as PEG,can be expressed as either a number-average molecular weight or aweight-average molecular weight. Unless otherwise indicated, allreferences to molecular weight herein refer to the weight-averagemolecular weight. Both molecular weight determinations, number-averageand weight-average, can be measured using gel permeation chromatographyor other liquid chromatography techniques. Other methods for measuringmolecular weight values can also be used, such as the use of end-groupanalysis or the measurement of colligative properties (e.g.,freezing-point depression, boiling-point elevation, or osmotic pressure)to determine number-average molecular weight or the use of lightscattering techniques, ultracentrifugation or viscometry to determineweight-average molecular weight. The polymers of the invention aretypically polydisperse (i.e., number-average molecular weight andweight-average molecular weight of the polymers are not equal),possessing low polydispersity values of preferably less than about 1.2,more preferably less than about 1.15, still more preferably less thanabout 1.10, yet still more preferably less than about 1.05, and mostpreferably less than about 1.03. As used herein, references will attimes be made to a single water-soluble polymer having either aweight-average molecular weight or number-average molecular weight; suchreferences will be understood to mean that the single-water solublepolymer was obtained from a composition of water-soluble polymers havingthe stated molecular weight.

The terms “active” or “activated” when used in conjunction with aparticular functional group, refer to a reactive functional group thatreacts readily with an electrophile or a nucleophile on anothermolecule. This is in contrast to those groups that require strongcatalysts or highly impractical reaction conditions in order to react(i.e., a “non-reactive” or “inert” group).

As used herein, the term “functional group” or any synonym thereof ismeant to encompass protected forms thereof as well as unprotected forms.

The terms “spacer moiety,” “linkage” or “linker” are used herein torefer to an atom or a collection of atoms used to link interconnectingmoieties such as a terminus of a polymer and an active agent or anelectrophile or nucleophile of an active agent. The spacer moiety may behydrolytically stable or may include a physiologically hydrolyzable orenzymatically degradable linkage.

“Alkyl” refers to a hydrocarbon chain, typically ranging from about 1 to15 atoms in length. Such hydrocarbon chains are preferably but notnecessarily saturated and may be branched or straight chain, althoughtypically straight chain is preferred. Exemplary alkyl groups includemethyl, ethyl, propyl, butyl, pentyl, 1-methylbutyl, 1-ethylpropyl,3-methylpentyl, and the like. As used herein, “alkyl” includescycloalkyl as well as cycloalkylene-containing alkyl.

“Lower alkyl” refers to an alkyl group containing from 1 to 6 carbonatoms, and may be straight chain or branched. Nonlimiting examples oflower alkyl include methyl, ethyl, n-butyl, i-butyl, and t-butyl.

“Cycloalkyl” refers to a saturated or unsaturated cyclic hydrocarbonchain, including bridged, fused, or spiro cyclic compounds, preferablymade up of 3 to about 12 carbon atoms, more preferably 3 to about 8carbon atoms. “Cycloalkylene” refers to a cycloalkyl group that isinserted into an alkyl chain by bonding of the chain at any two carbonsin the cyclic ring system.

“Alkoxy” refers to an —O—R group, wherein R is alkyl or substitutedalkyl, preferably C₁₋₆ alkyl (e.g., methoxy, ethoxy, propyloxy, and soforth).

The term “substituted” as in, for example, “substituted alkyl,” refersto a moiety (e.g., an alkyl group) substituted with one or morenoninterfering substituents, such as, but not limited to: alkyl, C₃₋₈cycloalkyl, e.g., cyclopropyl, cyclobutyl, and the like; halo, e.g.,fluoro, chloro, bromo, and iodo; cyano; alkoxy; lower phenyl;substituted phenyl; and the like. “Substituted aryl” is aryl having oneor more noninterfering substituents. For substitutions on a phenyl ring,the substituents may be in any orientation (i.e., ortho, meta, or para).

“Noninterfering substituents” are those groups that, when present in amolecule, are typically nonreactive with other functional groupscontained within the molecule.

“Aryl” means one or more aromatic rings, each of 5 or 6 core carbonatoms. Aryl includes multiple aryl rings that may be fused, as innaphthyl, or unfused, as in biphenyl. Aryl rings may also be fused orunfused with one or more cyclic hydrocarbon, heteroaryl, or heterocyclicrings. As used herein, “aryl” includes heteroaryl.

“Heteroaryl” is an aryl group containing from one to four heteroatoms,preferably sulfur, oxygen, or nitrogen, or a combination thereof.Heteroaryl rings may also be fused with one or more cyclic hydrocarbon,heterocyclic, aryl, or heteroaryl rings.

“Heterocycle” or “heterocyclic” means one or more rings of 5-12 atoms,preferably 5-7 atoms, with or without unsaturation or aromatic characterand having at least one ring atom that is not a carbon. Preferredheteroatoms include sulfur, oxygen, and nitrogen.

“Substituted heteroaryl” is heteroaryl having one or more noninterferinggroups as substituents.

“Substituted heterocycle” is a heterocycle having one or more sidechains formed from noninterfering substituents.

An “organic radical” as used herein shall include alkyl, substitutedalkyl, aryl and substituted aryl.

“Electrophile” and “electrophilic group” refer to an ion or atom orcollection of atoms, that may be ionic, having an electrophilic center,i.e., a center that is electron seeking, capable of reacting with anucleophile.

“Nucleophile” and “nucelophilic group” refers to an ion or atom orcollection of atoms that may be ionic having a nucleophilic center,i.e., a center that is seeking an electrophilic center or capable ofreacting with an electrophile.

A “physiologically cleavable” or “hydrolyzable” bond is a bond thatreacts with water (i.e., is hydrolyzed) under physiological conditions.Preferred are bonds that have a hydrolysis half-life at pH 8, 25° C. ofless than about 30 minutes. The tendency of a bond to hydrolyze in waterwill depend not only on the general type of linkage connecting two givenatoms but also on the substituents attached to these two given atoms.Appropriate hydrolytically unstable or weak linkages include but are notlimited to carboxylate ester, phosphate ester, anhydrides, acetals,ketals, acyloxyalkyl ether, imine, orthoester, peptide andoligonucleotide.

An “enzymatically degradable linkage” means a linkage that is subject todegradation by one or more enzymes.

A “hydrolytically stable” linkage or bond refers to a chemical bond,typically a covalent bond, that is substantially stable in water, thatis to say, does not undergo hydrolysis under physiological conditions toany appreciable extent over an extended period of time. Examples ofhydrolytically stable linkages include, but are not limited to, thefollowing: carbon-carbon bonds (e.g., in aliphatic chains), ethers,amides, urethane, and the like. Generally, a hydrolytically stablelinkage is one that exhibits a rate of hydrolysis of less than about1-2% per day under physiological conditions. Hydrolysis rates ofrepresentative chemical bonds can be found in most standard chemistrytextbooks.

“Pharmaceutically acceptable excipient” refers to an excipient that mayoptionally be included in a composition and that causes no significantadverse toxicological effects to a patient upon administration.

“Therapeutically effective amount” is used herein to mean the amount ofa conjugate that is needed to provide a desired level of the conjugate(or corresponding unconjugated active agent) in the bloodstream or inthe target tissue following administration. The precise amount willdepend upon numerous factors, e.g., the particular active agent, thecomponents and physical characteristics of the therapeutic composition,the intended patient population, the mode of delivery, individualpatient considerations, and the like, and can readily be determined byone skilled in the art.

“Multi-functional” means a polymer having three or more functionalgroups contained therein, where the functional groups may be the same ordifferent. Multi-functional polymeric reagents will typically contain anumber of functional groups in one or more of the following ranges: fromabout 3-100 functional groups; from 3-50 functional groups; from 3-25functional groups; from 3-15 functional groups; from 3 to 10 functionalgroups. Exemplary numbers of functional groups include 3, 4, 5, 6, 7, 8,9 and 10 functional groups within the polymer backbone.

“Optional” or “optionally” means that the subsequently describedcircumstance may or may not occur, so that the description includesinstances where the circumstance occurs and instances where it does not.

“Substantially” (unless specifically defined for a particular contextelsewhere or the context clearly dictates otherwise) means nearlytotally or completely, for instance, satisfying one or more of thefollowing: greater than 50%, 51% or greater, 75% or greater, 80% orgreater, 90% or greater, and 95% or greater of the condition.

The phrase “substantially nonaqueous conditions” means a composition orreaction medium having less than 10,000 parts per million of water (lessthan 1%), more preferably having less than 1,000 parts per million ofwater (less than 0.1%), still more preferably less than 100 parts permillion of water (less than 0.01%), still more preferably les than 10parts per million of water (less than 0.001%). Preferably, but notnecessarily, substantially nonaqueous conditions includes an inertatmosphere.

Unless the context clearly dictates otherwise, when the term “about”precedes a numerical value, the numerical value is understood to mean±10% of the stated numerical value.

Method For Preparing A Substituted Maleamic Acid-Terminated,Water-Soluble Polymer

In one or more embodiments of the present invention, a method forpreparing a substituted maleamic acid-terminated, water-soluble polymeris provided. The method comprises:

-   a) combining an amine-terminated, water-soluble polymer with a    maleimide reagent under substantially nonaqueous conditions to form    a substituted maleamic acid-terminated, water-soluble polymer; and-   a′) optionally, isolating the substituted maleamic acid-terminated,    water-soluble polymer.

The combining step requires, as a starting material, anamine-terminated, water-soluble polymer. As used herein, an“amine-terminated, water-soluble polymer” is any water-soluble polymerthat bears at least one amine group (“—NH₂”), regardless of whether theamine group is actually located at a terminus of the water-solublepolymer. Typically, although not necessarily, the amine-terminated,water-soluble polymer will have only one amine group, but theamine-terminated, water-soluble polymer can have more than one aminegroup. Thus, the amine-terminated, water-soluble polymer can (forexample) have a total number of amine groups of any one of one, two,three, four, five, six, seven, eight, nine and ten.

An exemplary amine-terminated, water-soluble polymer comprises thefollowing structure:POLY—(X²)_(c)—NH₂  (Formula II)wherein:

POLY is a water-soluble polymer (preferably linear or branched, andpreferably is CH₃O—(CH₂CH₂O)_(n)—CH₂CH₂—, wherein (n) is 2 to 4000 whenPOLY is linear);

(c) is zero or one (preferably zero); and

X², wherein present, is a spacer moiety.

The combining step also requires a maleimide reagent. The maleimidereagent is a reagent that—upon combination with an amine-terminated,water-soluble polymer—will result in the formation of one of thefollowing: a maleimide-terminated, water-soluble polymer; or asubstituted maleamic acid-terminated, water-soluble polymer.

An exemplary maleimide reagent comprises the following structure:

wherein:

Y¹ is O or S (preferably O);

Y² is O or S (preferably O);

(a) is an integer from 1 to 20 (preferably one or two);

R¹, in each instance, is independently H or an organic radical(preferably H);

R², in each instance, is independently H or an organic radical(preferably H);

R³, in each instance, is independently H or an organic radical(preferably H); and

R⁴, in each instance, is independently H or an organic radical(preferably H).

A preferred maleimide reagent is an N-alkoxycarbonylmaleimide,particularly where alkoxy is lower alkoxy. A preferredN-alkoxycarbonylmaleimide, N-methoxycarbonylmaleimide, is shown below:

The combining step includes bringing the amine-terminated, water-solublepolymer in contact with the maleimide reagent and can be accomplished inany method known to those of ordinary skill in the art. For example, acomposition comprising the amine-terminated, water-soluble polymer and acomposition comprising the maleimide reagent can be combined in areaction vessel. The combining step is, however, carried out to minimizethe introduction of water.

Following the combining step, the method for preparing a substitutedmaleamic acid-terminated, water-soluble polymer, optionally includes thestep of isolating the substituted maleamic acid-terminated,water-soluble polymer.

When it is intended to isolate the substituted maleamic acid-terminated,water-soluble polymer (and thus carry out the optional step of isolatingthe substituted maleamic acid-terminated, water-soluble polymer), anyart-known technique can be used to isolate the substituted maleamicacid-terminated, water-soluble polymer and the invention is not limitedin this regard. For example, isolation techniques selected from thegroup consisting of chromatography (e.g., silica-gel chromatography,HPLC chromatography, affinity-based chromatography, ion-exchangechromatography, and so forth), electrophoresis, precipitation(including, for example, recrystallization) and extraction can be usedto isolate the substituted maleamic acid-terminated, water-solublepolymer. A preferred isolation technique is precipitation which can beaccomplished using art-known methods (such as adding an excess ofisopropyl alcohol, diethyl ether, MTBE, heptane, THF, hexane, and soforth, to cause the product to precipitate). Precipitation techniqueswill yield a dried substituted maleamic acid-terminated, water-solublepolymer. Other techniques can also be used to result in the driedsubstituted maleamic acid-terminated, water-soluble polymer.

The substituted maleamic acid-terminated, water-soluble polymer can takeany number of forms. A preferred form is a substituted maleamicacid-terminated, water-soluble polymer comprising the following thefollowing structure:

wherein:

POLY is a water-soluble polymer;

(b) is zero or one;

X¹, wherein present, is a spacer moiety;

Y¹ is O or S;

Y² is O or S;

(a) is an integer from 1 to 20;

R¹, in each instance, is independently H or an organic radical;

R², in each instance, is independently H or an organic radical;

R³, in each instance, is independently H or an organic radical; and

R⁴, in each instance, is independently H or an organic radical.

It is preferred that a substituted maleamic acid-terminated,water-soluble polymer is provided in isolated form, meaning acomposition wherein at least about 70% (more preferably at least 80%,and most preferably at least 90%) of all polymer species in thecomposition is in the substituted maleamic acid-terminated,water-soluble polymer form (and not in the amine-terminated,water-soluble form or the maleimide-terminated, water-soluble polymerform).

Before using any isolated substituted maleamic acid-terminated,water-soluble polymer, it is typical to carry out the additional step ofredissolving the isolated (and typically dried) substituted maleamicacid-terminated, water-soluble polymer to regenerate the maleamicacid-terminated, water-soluble polymer in a nonaqueous liquid system.

The steps of the method used to prepare a maleimide-terminated,water-soluble polymer are typically carried out in an organic solvent.Although any organic solvent can be used and the invention is notlimited in this regard, exemplary organic solvents include thosesolvents selected from the group consisting of halogenated aliphatichydrocarbons, alcohols, aromatic hydrocarbons, alcohols, halogenatedaromatic hydrocarbons, amides (including DMF), nitrites (includingacetonitriles), ketones (including acetone), acetates (including ethylacetate), ethers, cyclic ethers, and combinations thereof. Examples ofpreferred organic solvents include those selected from the groupconsisting of methylene chloride (or dichloromethane), chloroform,octanol, toluene, methyl t-butyl ether, THF (tetrahydrofuran), ethylacetate, diethylcarbonate, acetone, acetonitrile, DMF (dimethylformamide), DMSO, dimethylacetamide, N-cyclohexylpyrrolidinone,cyclohexane and combinations thereof.

The method for preparing a substituted maleamic acid-terminated,water-soluble polymer has utility as, among other things, providing anintermediate that is useful in the formation of a maleimide-terminated,water-soluble polymer (as will be discussed herein). By performing thismethod, it is possible to provide greater, reproducible yields of thesubstituted maleamic acid-terminated, water-soluble polymer from theamine-terminated, water-soluble polymer, thereby providing a more pureintermediate that can result in a more pure maleimide-terminated polymercomposition and corresponding conjugate composition formed therefrom.Furthermore, the method provides for compositions that have lessmaleamic acid-based impurities in the composition.

Method For Preparing a Maleimide-Terminated, Water-Soluble Polymer

In one or more embodiments of the invention, a method for preparing amaleimide-terminated, water-soluble polymer is provided, the methodcomprising

-   a) combining an amine-terminated, water-soluble polymer with a    maleimide reagent under substantially nonaqueous conditions to form    a substituted maleamic acid-terminated, water-soluble polymer; and-   b) exposing the maleamic acid-terminated, water-soluble polymer to    elimination conditions under substantially nonaqueous conditions to    thereby result in a maleimide-terminated, water-soluble polymer.

The step of combining an amine-terminated, water-soluble polymer with amaleimide reagent under substantially nonaqueous conditions to form asubstituted maleamic acid-terminated, water-soluble polymer can becarried out as described above with respect to the method for preparinga substituted maleamic acid-terminated, water-soluble polymer.

Once the combining step has been carried out, the present method forpreparing a maleimide-terminated, water-soluble polymer also includesthe optional steps of isolating and redissolving the substitutedmaleamic acid-terminated, water-soluble polymer. Each of these optionalsteps (the optional isolating step and optional redissolving step) canbe carried out as described above with respect to the method forpreparing a substituted maleamic acid-terminated, water-soluble polymer.

The steps of the present method for preparing a maleimide-terminated,water-soluble polymer include the step of exposing the maleamicacid-terminated, water-soluble polymer to elimination conditionscomprises heating the maleamic acid-terminated, water-soluble polymer.Any art-known elimination conditions can be used and the invention isnot limited in this regard. For example, suitable elimination conditionscomprise refluxing the acid-terminated, water-soluble polymer at atemperature of greater than at least about 35° C., more preferably atleast about 40° C.

Exposure to elimination conditions can also include removing water fromthe reaction medium, by for example, exposing the maleamicacid-terminated, water-soluble polymer to a drying agent (such as addingNaHCO₃, Na₂CO₃, CaCl₂, CaSO₄, MgSO₄, KOH, Na₂SO₄, K₂CO₃, KHCO₃ andcombinations thereof), a molecular sieve (e.g., aluminum silicates),azeotropic distillation and combinations of any of the foregoing.

Catalysts can also be used to enhance the kinetics of the method. Inthis regard it is preferred to carry out the present method forpreparing a maleimide-terminated, water-soluble polymer in the presenceof a catalyst such as a non-nucleophilic amine catalyst or a basiccatalyst. With regard to non-nucleophilic amine catalysts, stericallyhindered non-nucleophilic amine catalysts are preferred. Examples ofnon-nucleophilic amine catalysts include those selected from the groupconsisting of DMAP (N,N-dimethyl-4-aminopyridine), DBU(1,8-diazabicyclo[5.4.0]undec-7-ene), DABCO(1,4-diazabicyclo[2.2.2]octane), diisopropylethylamine, triethylamine,n-methyl morpholine. Examples of sterically hindered non-nucleophilicamine catalysts include DMAP (N,N-dimethyl-4-aminopyridine), DBU(1,8-diazabicyclo[5.4.0]undec-7-ene), DABCO(1,4-diazabicyclo[2.2.2]octane), and diisopropylethylamine. Examples ofbasic catalysts include sodium carbonate, sodium bicarbonate, potassiumcarbonate and potassium bicarbonate.

The steps of the method used to prepare a maleimide-terminated,water-soluble polymer are typically carried out in an organic solvent.Although any organic solvent can be used and the invention is notlimited in this regard, exemplary organic solvents include thosesolvents selected from the group consisting of halogenated aliphatichydrocarbons, alcohols, aromatic hydrocarbons, alcohols, halogenatedaromatic hydrocarbons, amides (including DMF), nitrites (includingacetonitriles), ketones (including acetone), acetates (including ethylacetate), ethers, cyclic ethers, and combinations thereof. Examples ofpreferred organic solvents include those selected from the groupconsisting of methylene chloride (or dichloromethane), chloroform,octanol, toluene, methyl t-butyl ether, THF, ethyl acetate,diethylcarbonate, acetone, acetonitrile, DMF, DMSO, dimethylacetamide,N-cyclohexylpyrrolidinone, cyclohexane and combinations thereof.

The maleimide-terminated, water-soluble polymer can have a variety ofstructures and will depend upon the structure of the substitutedmaleamic acid-terminated, water-soluble polymer from which it derives.An exemplary, maleimide-terminated, water-soluble polymer prepared inaccordance with the presence method will be of the following structure:

wherein each of POLY, X² and (c) are defined as provided in Formula IIand each of R³ and R⁴ are defined as provided in Formula III.

A particularly preferred maleimide-terminated, water-soluble polymerwill comprise the following structure:

wherein (n) is an integer from 2 to about 4000.

Maleimide-Terminated, Water-Soluble Polymer-Containing Composition

In one or more embodiments of the invention, a maleimide-terminated,water-soluble polymer-containing composition is provided, thecomposition resulting from the method comprising:

a) combining an amine-terminated, water-soluble polymer with a maleimidereagent under substantially nonaqueous conditions to form a maleamicacid-terminated, water-soluble polymer; and

b) exposing the maleamic acid-terminated, water-soluble polymer toelimination conditions under substantially nonaqueous conditions tothereby result in a maleimide-terminated, water-solublepolymer-containing composition.

Thus, included within the invention are compositions ofmaleimide-terminated, water-soluble polymers formed in accordance withthe method provided. The compositions resulting from the method arebelieved to have greater purity than previously known methods.Specifically, the maleimide-terminated, water-soluble polymer-containingcompositions possess relatively low percentages of maleamic acidterminated, water-soluble polymers (e.g., typically less than fourpercent and often less than two percent). In addition, themaleimide-terminated, water-soluble polymer-containing compositions aresubstantially free of furan, preferably completely free of furan.

In another embodiment of the invention, maleimide-terminated,water-soluble polymer-containing compositions are provided, suchcompositions comprising polymeric species wherein at least 70% of thepolymeric species in the composition are maleimide-terminated,water-soluble polymers and further wherein the composition comprisesopen ring ester polymeric species. The open ring ester polymeric specieshas the structure

wherein each of POLY, X¹, (a), (b), R¹, R², R³, R⁴, Y¹ and Y² is asdefined with respect to Formula I. This “open ring ester” is not foundin connection with the aqueous-based N-alkoxycarbonylmaleimide route forpreparing maleimide-terminated, water-soluble polymers.

Method for Preparing a Conjugate-Containing Composition

In one or more embodiments of the invention, a method for preparing aconjugate-containing composition is provided, the method comprisingcombining (in a reaction vessel) a thiol-containing biologically activeagent (such as a cysteine-containing protein or polypeptide) with amaleimide-terminated, water-soluble polymer composition as providedherein to thereby result in a conjugate-containing composition. Althoughapproaches for conjugating maleimide-terminated, water-soluble polymersto thiol-containing biologically active agents have been described, anexemplary approach involves dissolving the maleimide-terminated,water-soluble polymer in deionized water to make a 10% reagent solutionand combining with a thiol-containing biologically active agent (at afive- to twenty-fold molar excess the polymer to the thiol-containingbiologically active agent) and mixing well. After about one hour ofreaction at room temperature, the reaction vial can cooled and mixed forabout twelve hours to ensure sufficient reaction time. The pH of thereaction can be conducted at about 7.

Thus, included within the invention are methods for preparingconjugate-containing compositions using the inventivemaleimide-terminated, water-soluble polymer compositions of theinvention. The thiol-containing active agent can be any protein bearinga cysteine residue that is not involved in intraprotein disulfidebinding.

Conjugate-Containing Compositions

In one or more embodiments of the invention, a conjugate-containingcomposition is provided, the composition resulting from the methodcomprising combining a thiol-containing active agent with amaleimide-terminated, water-soluble polymer-containing composition asprovided herein.

Thus, included within the invention are conjugate-containingcompositions formed in accordance with the provided method for preparingconjugate-containing compositions. The compositions resulting from themethod are believed to have greater purity than previously knownmethods. The conjugate-containing compositions, like themaleimide-terminated, water soluble polymer compositions used to createthem, possess relatively low percentages of maleamic acid terminated,water-soluble polymers (e.g., typically less than four percent and oftenless than two percent). In addition, the conjugate-containingcompositions are substantially free of furan, preferably completely freeof furan.

The Water-Soluble Polymer (“POLY”)

As used herein, the term “water-soluble polymer” includes those watersoluble polymers that are biocompatible and nonimmunogenic andspecifically excludes any water soluble polymer segments that are notbiocompatible and nonimmunogenic. With respect to biocompatibility, asubstance is considered biocompatible if the beneficial effectsassociated with use of the substance alone or with another substance(e.g., active agent) in connection with living tissues (e.g.,administration to a patient) outweighs any deleterious effects asevaluated by a clinician, e.g., a physician. With respect tononimmunogenicity, a substance is considered nonimmunogenic if theintended use of the substance in vivo does not produce an undesiredimmune response (e.g., the formation of antibodies) or, if an immuneresponse is produced, that such a response is not deemed clinicallysignificant or important as evaluated by a clinician. It is particularlypreferred that the water soluble polymer segments described herein aswell as conjugates are biocompatible and nonimmunogenic.

When referring to the polymer, it is to be understood that the polymercan be any of a number of water soluble and non-peptidic polymers, suchas those described herein as suitable for use in the present invention.Preferably, poly(ethylene glycol) (i.e., PEG) is the polymer. The termPEG includes poly(ethylene glycol) in any of a number of geometries orforms, including linear forms, branched or multi-arm forms (e.g., forkedPEG or PEG attached to a polyol core), pendant PEG, or PEG withdegradable linkages therein, to be more fully described below.

The number of functional groups carried by the polymer and the positionof the functional groups may vary. Typically, the polymer will comprise1 to about 25 functional groups, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10 functional groups. Linear polymers, such as PEG polymers, willtypically comprise one or two functional groups positioned at theterminus of the polymer chain. If the PEG polymer is monofunctional(i.e., linear mPEG), the polymer will include a single functional group.If the PEG polymer is difunctional, the polymer may contain twoindependently selected functional groups, one at each terminus of thepolymer chain. As would be understood, multi-arm or branched polymersmay comprise a greater number of functional groups.

Multi-armed or branched PEG molecules, such as those described in U.S.Pat. No. 5,932,462, which is incorporated by reference herein in itsentirety, can also be used as the PEG polymer. Generally speaking, amulti-armed or branched polymer possesses two or more polymer “arms”extending from a central branch point. For example, an exemplarybranched PEG polymer has the structure:

wherein PEG₁ and PEG₂ are PEG polymers in any of the forms or geometriesdescribed herein, and which can be the same or different, and L′ is ahydrolytically stable linkage. An exemplary branched PEG has thestructure:

wherein poly_(a) and poly_(b) are PEG backbones, such as methoxypoly(ethylene glycol); R″ is a nonreactive moiety, such as H, methyl ora PEG backbone; and P and Q are nonreactive linkages. In a preferredembodiment, the branched PEG polymer is methoxy poly(ethylene glycol)disubstituted lysine.

The branched PEG structure can be attached to a third oligomer orpolymer chain as shown below:

wherein PEG₃ is a third PEG oligomer or polymer chain, which can be thesame or different from PEG₁ and PEG₂.

The PEG polymer can alternatively comprise a forked PEG. Generallyspeaking, a polymer having a forked structure is characterized as havinga polymer chain attached to two or more functional groups via covalentlinkages extending from a hydrolytically stable branch point in thepolymer. An example of a forked PEG is represented by PEG—YCHZ₂, where Yis a linking group and Z is an activated terminal group for covalentattachment to a biologically active agent. The Z group is linked to CHby a chain of atoms of defined length. U.S. Pat. No. 6,362,254, thecontents of which are incorporated by reference herein, disclosesvarious forked PEG structures capable of use in the present invention.The chain of atoms linking the Z functional groups (e.g., hydroxylgroups) to the branching carbon atom serve as a tethering group and maycomprise, for example, an alkyl chain, ether linkage, ester linkage,amide linkage, or combinations thereof.

The PEG polymer may comprise a pendant PEG molecule having reactivegroups (e.g., hydroxyl groups) covalently attached along the length ofthe PEG backbone rather than at the end of the PEG chain. The pendantreactive groups can be attached to the PEG backbone directly or througha linking moiety, such as an alkylene group.

In addition to the above-described forms of PEG, the polymer can also beprepared with one or more hydrolytically stable or degradable linkagesin the polymer backbone, including any of the above described polymers.For example, PEG can be prepared with ester linkages in the polymerbackbone that are subject to hydrolysis. As shown below, this hydrolysisresults in cleavage of the polymer into fragments of lower molecularweight:

Other hydrolytically degradable linkages, useful as a degradable linkagewithin a polymer backbone, include carbonate linkages; imine linkagesresulting, for example, from reaction of an amine and an aldehyde (see,e.g., Ouchi et al., Polymer Preprints, 38(1):582-3 (1997), which isincorporated herein by reference.); phosphate ester linkages formed, forexample, by reacting an alcohol with a phosphate group; hydrazonelinkages which are typically formed by reaction of a hydrazide and analdehyde; acetal linkages that are typically formed by reaction betweenan aldehyde and an alcohol; ortho ester linkages that are, for example,formed by reaction between acid derivatives and an alcohol; andoligonucleotide linkages formed by, for example, a phosphoramiditegroup, e.g., at the end of a polymer, and a 5′ hydroxyl group of anoligonucleotide. The use of many of the above-described degradablelinkages is less preferred due to nucleophilic reactivity of many of theunstable linkages with amine groups.

It is understood by those skilled in the art that the term poly(ethyleneglycol) or PEG represents or includes all the above forms of PEG.

Any of a variety of other polymers comprising other non-peptidic andwater soluble polymer chains can also be used in the present invention.The polymer can be linear, or can be in any of the above-described forms(e.g., branched, forked, and the like). Examples of suitable polymersinclude, but are not limited to, other poly(alkylene glycols),copolymers of ethylene glycol and propylene glycol, poly(olefinicalcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide),poly(hydroxyalkylmethacrylate), poly(saccharides), poly(α-hydroxyaceticacid), poly(acrylic acid), poly(vinyl alcohol), polyphosphazene,polyoxazolines, poly(N-acryloylmorpholine), such as described in U.S.Pat. No. 5,629,384, which is incorporated by reference herein in itsentirety, and copolymers, terpolymers, and mixtures thereof.

Although the molecular weight of the water soluble polymer can varydepending on the desired application, the configuration of the polymerstructure, the degree of branching, and the like, the molecular weightwill satisfy one or more of the following values: greater than 100Daltons; greater than 200 Daltons; greater than 400 Daltons; greaterthan 500 Daltons, greater than 750 Daltons; greater than 900 Daltons;greater than 1,000 Daltons, greater than 1,400 Daltons; greater than1,500 Daltons, greater than 1,900 Daltons; greater than 2,000 Daltons;greater than 2,200 Daltons; greater than 2,500 Daltons; greater than3,000 Daltons; greater than 4,000 Daltons; greater than 4,900 Daltons;greater than 5,000 Daltons; greater than 6,000 Daltons; greater than7,000 Daltons; greater than 7,500 Daltons, greater than 9,000 Daltons;greater than 10,000 Daltons; greater than 11,000 Daltons; greater than14,000 Daltons, greater than 15,000 Daltons; greater than 16,000Daltons; greater than 19,000 Daltons; greater than 20,000 Daltons;greater than 21,000 Daltons; greater than 22,000 Daltons, greater than25,000 Daltons; and greater than 30,000 Daltons. It is understood thatthe maximum limit of molecular weight for any given water solublepolymer segment useful herein is less than about 300,000 Daltons.

The molecular weight of the polymer will typically fall into at leastone of the following ranges: from about 100 Daltons to about 100,000Daltons; from about 200 Daltons to about 60,000 Daltons; from about 300Daltons to about 40,000 Daltons.

Exemplary molecular weights for the water soluble polymer include about100 Daltons, about 200 Daltons, about 300 Daltons, about 350 Daltons,about 400 Daltons, about 500 Daltons, about 550 Daltons, about 600Daltons, about 700 Daltons, about 750 Daltons, about 800 Daltons, about900 Daltons, about 1,000 Daltons, about 2,000 Daltons, about 2,200Daltons, about 2,500 Daltons, about 3,000 Daltons, about 4,000 Daltons,about 4,400 Daltons, about 5,000 Daltons, about 6,000 Daltons, about7,000 Daltons, about 7,500 Daltons, about 8,000 Daltons, about 9,000Daltons, about 10,000 Daltons, about 11,000 Daltons, about 12,000Daltons, about 13,000 Daltons, about 14,000 Daltons, about 15,000Daltons, about 20,000 Daltons, about 22,500 Daltons, about 25,000Daltons, about 30,000 Daltons, about 35,000 Daltons, about 40,000Daltons, about 50,000 Daltons, about 60,000 Daltons, and about 75,000Daltons.

With respect to branched versions of the polymer, exemplary ranges ofsuitable sizes for the total molecular weight of the polymer (as basedessentially on the combined weights of the two water soluble polymerportions) include the following: from about 200 Daltons to about 100,000Daltons; from about 1,000 Daltons to about 80,000 Daltons; from about2,000 Daltons to about 50,000 Daltons; from about 4,000 Daltons to about25,000 Daltons; and from about 10,000 Daltons to about 40,000 Daltons.More particularly, total weight average molecular weight of a branchedversion of the polymer of the invention corresponds to one of thefollowing: 400; 1,000; 1,500; 2,000; 3000; 4,000; 10,000; 15,000;20,000; 30,000; 40,000; 50,000; 60,000; or 80,000.

With respect to PEG, wherein a structure comprising a repeating ethyleneoxide monomer, such as “—(CH₂CH₂O)_(n)—” or “—(OCH₂CH₂)_(n)” can beprovided, preferred values for (n) include: from about 3 to about 3,000;from about 10 to about 3,000; from about 15 to about 3,000; from about20 to about 3,000; from about 25 to about 3,000; from about 30 to about3,000; from about 40 to about 3,000; from about 50 to about 3,000; fromabout 55 to about 3,000; from about 75 to about 3,000; from about 100 toabout 3,000; and from about 225 to about 3,000.

The Spacer Moiety (“X¹”, “X²”, and so forth)

Optionally, a spacer moiety can link the water-soluble polymer to themaleimide and/or from the maleimidyl moiety to the residue of athiol-containing active agent. Exemplary spacer moieties include thefollowing: —O—, —S—, —C(O)—, —O—C(O)—, —C(O)—O—, —C(O)—NH—,—NH—C(O)—NH—, —O—C(O)—NH—, —C(S)—, —CH₂—, —CH₂—CH₂—, —CH₂—CH₂—CH₂—,—CH₂—CH₂—CH₂—CH₂—, —O—CH₂—, —CH₂—O—, —O—CH₂—CH₂—, —CH₂—O—CH₂—,—CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—, —CH₂—O—CH₂—CH₂—, —CH₂—CH₂—O—CH₂—,—CH₂—CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—CH₂—, —CH₂—O—CH₂—CH₂—CH₂—,—CH₂—CH₂—O—CH₂—CH₂—, —CH₂—CH₂—CH₂—O—CH₂—, —CH₂—CH₂—CH₂—CH₂—O—,—C(O)—NH—CH₂—, —C(O)—NH—CH₂—CH₂—, —CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—C(O)—NH—,—C(O)—NH—CH₂—CH₂—CH₂—, —CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—C(O)—NH—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—, —C(O)—NH—CH₂—CH₂—CH₂—CH₂—,—CH₂—C(O)—NH—CH₂—CH₂—CH₂—, —CH₂—CH₂—C(O)—NH—CH₂—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—,—CH₂—CH₂—CH₂—CH₂—C(O)—NH—, —C(O)—O—CH₂—, —CH₂—C(O)—O—CH₂—,—CH₂—CH₂—C(O)—O—CH₂—, —C(O)—O—CH₂—CH₂—, —NH—C(O)—CH₂—,—CH₂—NH—C(O)—CH₂—, —CH₂—CH₂—NH—C(O)—CH₂—, —NH—C(O)—CH₂—CH₂—,—CH₂—NH—C(O)—CH₂—CH₂—, —CH₂—CH₂—NH—C(O)—CH₂—CH₂—, —C(O)—NH—CH₂—,—C(O)—NH—CH₂—CH₂—, —O—C(O)—NH—CH₂—, —O—C(O)—NH—CH₂—CH₂—,—O—C(O)—NH—CH₂—CH₂—CH₂—, —NH—CH₂—, —NH—CH₂—CH₂—, —CH₂—NH—CH₂—,—CH₂—CH₂—NH—CH₂—, —C(O)—CH₂—, —C(O)—CH₂—CH₂—, —CH₂—C(O)—CH₂—,—CH₂—CH₂—C(O)—CH₂—, —CH₂—CH₂—C(O)—CH₂—CH₂—, —CH₂—CH₂—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—CH₂—,—O—C(O)—NH—[CH₂]₀₋₆—(OCH₂CH₂)₀₋₂—, —C(O)—NH—(CH₂)₁₋₆—NH—C(O)—,—NH—C(O)—NH—(CH₂)₁₋₆—NH—C(O)—, —O—C(O)—CH₂—, —O—C(O)—CH₂—CH₂—,—O—C(O)—CH₂—CH₂—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—, bivalent cycloalkyl group,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—CH₂—,O—C(O)—NH—[CH₂]_(f)—(OCH₂CH₂)_(n−), and combinations of two or more ofany of the foregoing, wherein (f) is 0 to 6, (n) is 0 to 20 (preferably0 to 10, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and more preferably4). In addition, each of the foregoing carbon-containing spacer moietiescan have a branched alkyl group attached thereto. Nonlimiting examplesof bivalent cycloalkyl (e.g., cycloalkylene) groups include C₃₋₈cycloalkyl, such as various isomers of cyclopropadiyl (e.g., 1,1-,cis-11,2-, or trans-1,2-cyclopropylene), cyclobutadiyl, cyclopentadiyl,cyclohexadiyl, and cycloheptadiyl. The cycloalkylene group can besubstituted with one or more alkyl groups, preferably C₁-C₆ alkylgroups.

Biologically Active Conjugates

The present invention also includes stabilized biologically activeconjugates comprising a nucleophilic biologically active moleculecapable of Michael addition covalently attached to the reactive polymerthrough a succinimide ring linkage. The biologically active agent ispreferably a protein bearing a thiol or amino group.

Suitable biologically active agents may be selected from, for example,hypnotics and sedatives, psychic energizers, tranquilizers, respiratorydrugs, anticonvulsants, muscle relaxants, antiparkinson agents (dopamineantagnonists), analgesics, anti-inflammatories, antianxiety drugs(anxiolytics), appetite suppressants, antimigraine agents, musclecontractants, anti-infectives (antibiotics, antivirals, antifungals,vaccines) antiarthritics, antimalarials, antiemetics, anepileptics,bronchodilators, cytokines, growth factors, anti-cancer agents,antithrombotic agents, antihypertensives, cardiovascular drugs,antiarrhythmics, antioxicants, anti-asthma agents, hormonal agentsincluding contraceptives, sympathomimetics, diuretics, lipid regulatingagents, antiandrogenic agents, antiparasitics, anticoagulants,neoplastics, antineoplastics, hypoglycemics, nutritional agents andsupplements, growth supplements, antienteritis agents, vaccines,antibodies, diagnostic agents, and contrasting agents.

Examples of active agents suitable for use in covalent attachment to thereactive polymer of the invention include, but are not limited to,calcitonin, erythropoietin (EPO), Factor VIII, Factor IX, ceredase,cerezyme, cyclosporin, granulocyte colony stimulating factor (GCSF),thrombopoietin (TPO), alpha-1 proteinase inhibitor, elcatonin,granulocyte macrophage colony stimulating factor (GMCSF), growthhormone, human growth hormone (HGH), growth hormone releasing hormone(GHRH), heparin, low molecular weight heparin (LMWH), interferon alpha,interferon beta, interferon gamma, interleukin-1 receptor,interleukin-2, interleukin-1 receptor antagonist, interleukin-3,interleukin-4, interleukin-6, luteinizing hormone releasing hormone(LHRH), factor IX insulin, pro-insulin, insulin analogues (e.g.,mono-acylated insulin as described in U.S. Pat. No. 5,922,675), amylin,C-peptide, somatostatin, somatostatin analogs including octreotide,vasopressin, follicle stimulating hormone (FSH), insulin-like growthfactor (IGF), insulintropin, macrophage colony stimulating factor(M-CSF), nerve growth factor (NGF), tissue growth factors, keratinocytegrowth factor (KGF), glial growth factor (GGF), tumor necrosis factor(TNF), endothelial growth factors, parathyroid hormone (PTH),glucagon-like peptide thymosin alpha 1, IIb/IIIa inhibitor, alpha-1antitrypsin, phosphodiesterase (PDE) compounds, VLA-4 inhibitors,bisphosphonates, respiratory syncytial virus antibody, cystic fibrosistransmembrane regulator (CFTR) gene, deoxyreibonuclease (Dnase),bactericidal/permeability increasing protein (BPI), anti-CMV antibody,13-cis retinoic acid, macrolides such as erythromycin, oleandomycin,troleandomycin, roxithromycin, clarithromycin, davercin, azithromycin,flurithromycin, dirithromycin, josamycin, spiromycin, midecamycin,leucomycin, miocamycin, rokitamycin, andazithromycin, and swinolide A;fluoroquinolones such as ciprofloxacin, ofloxacin, levofloxacin,trovafloxacin, alatrofloxacin, moxifloxicin, norfloxacin, enoxacin,grepafloxacin, gatifloxacin, lomefloxacin, sparfloxacin, temafloxacin,pefloxacin, amifloxacin, fleroxacin, tosufloxacin, prulifloxacin,irloxacin, pazufloxacin, clinafloxacin, and sitafloxacin,aminoglycosides such as gentamicin, netilmicin, paramecin, tobramycin,amikacin, kanamycin, neomycin, and streptomycin, vancomycin,teicoplanin, rampolanin, mideplanin, colistin, daptomycin, gramicidin,colistimethate, polymixins such as polymixin B, capreomycin, bacitracin,penems; penicillins including penicllinase-sensitive agents likepenicillin G, penicillin V, penicllinase-resistant agents likemethicillin, oxacillin, cloxacillin, dicloxacillin, floxacillin,nafcillin; gram negative microorganism active agents like ampicillin,amoxicillin, and hetacillin, cillin, and galampicillin; antipseudomonalpenicillins like carbenicillin, ticarcillin, azlocillin, mezlocillin,and piperacillin; cephalosporins like cefpodoxime, cefprozil, ceftbuten,ceftizoxime, ceftriaxone, cephalothin, cephapirin, cephalexin,cephradrine, cefoxitin, cefamandole, cefazolin, cephaloridine, cefaclor,cefadroxil, cephaloglycin, cefuroxime, ceforanide, cefotaxime,cefatrizine, cephacetrile, cefepime, cefixime, cefonicid, cefoperazone,cefotetan, cefmetazole, ceftazidime, loracarbef, and moxalactam,monobactams like aztreonam; and carbapenems such as imipenem, meropenem,pentamidine isethiouate, albuterol sulfate, lidocaine, metaproterenolsulfate, beclomethasone diprepionate, triamcinolone acetamide,budesonide acetonide, fluticasone, ipratropium bromide, flunisolide,cromolyn sodium, ergotamine tartrate and where applicable, analogues,agonists, antagonists, inhibitors, and pharmaceutically acceptable saltforms of the above. In reference to peptides and proteins, the inventionis intended to encompass synthetic, native, glycosylated,unglycosylated, pegylated forms, and biologically active fragments andanalogs thereof.

The present invention also includes pharmaceutical preparationscomprising a conjugate as provided herein in combination with apharmaceutical excipient. Generally, the conjugate itself will be in asolid form (e.g., a precipitate), which can be combined with a suitablepharmaceutical excipient that can be in either solid or liquid form.

Exemplary excipients include, without limitation, those selected fromthe group consisting of carbohydrates, inorganic salts, antimicrobialagents, antioxidants, surfactants, buffers, acids, bases, andcombinations thereof.

A carbohydrate such as a sugar, a derivatized sugar such as an alditol,aldonic acid, an esterified sugar, and/or a sugar polymer may be presentas an excipient. Specific carbohydrate excipients include, for example:monosaccharides, such as fructose, maltose, galactose, glucose,D-mannose, sorbose, and the like; disaccharides, such as lactose,sucrose, trehalose, cellobiose, and the like; polysaccharides, such asraffinose, melezitose, maltodextrins, dextrans, starches, and the like;and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol,sorbitol (glucitol), pyranosyl sorbitol, myoinositol, and the like.

The excipient can also include an inorganic salt or buffer such ascitric acid, sodium chloride, potassium chloride, sodium sulfate,potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic,and combinations thereof.

The preparation can also include an antimicrobial agent for preventingor deterring microbial growth. Nonlimiting examples of antimicrobialagents suitable for the present invention include benzalkonium chloride,benzethonium chloride, benzyl alcohol, cetylpyridinium chloride,chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate,thimersol, and combinations thereof.

An antioxidant can be present in the preparation as well. Antioxidantsare used to prevent oxidation, thereby preventing the deterioration ofthe conjugate or other components of the preparation. Suitableantioxidants for use in the present invention include, for example,ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene,hypophosphorous acid, monothioglycerol, propyl gallate, sodiumbisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, andcombinations thereof.

A surfactant can be present as an excipient. Exemplary surfactantsinclude: polysorbates, such as “Tween 20” and “Tween 80,” and pluronicssuch as F68 and F88 (both of which are available from BASF, Mount Olive,N.J.); sorbitan esters; lipids, such as phospholipids such as lecithinand other phosphatidylcholines, phosphatidylethanolamines (althoughpreferably not in liposomal form), fatty acids and fatty esters;steroids, such as cholesterol; and chelating agents, such as EDTA, zincand other such suitable cations.

Acids or bases can be present as an excipient in the preparation.Nonlimiting examples of acids that can be used include those acidsselected from the group consisting of hydrochloric acid, acetic acid,phosphoric acid, citric acid, malic acid, lactic acid, formic acid,trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid,sulfuric acid, fumaric acid, and combinations thereof. Examples ofsuitable bases include, without limitation, bases selected from thegroup consisting of sodium hydroxide, sodium acetate, ammoniumhydroxide, potassium hydroxide, ammonium acetate, potassium acetate,sodium phosphate, potassium phosphate, sodium citrate, sodium formate,sodium sulfate, potassium sulfate, potassium fumerate, and combinationsthereof.

The pharmaceutical preparations encompass all types of formulations andin particular those that are suited for injection, e.g., powders thatcan be reconstituted as well as suspensions and solutions. The amount ofthe conjugate (i.e., the conjugate formed between the active agent andthe polymer described herein) in the composition will vary depending ona number of factors, but will optimally be a therapeutically effectivedose when the composition is stored in a unit dose container (e.g., avial). In addition, the pharmaceutical preparation can be housed in asyringe. A therapeutically effective dose can be determinedexperimentally by repeated administration of increasing amounts of theconjugate in order to determine which amount produces a clinicallydesired endpoint.

The amount of any individual excipient in the composition will varydepending on the activity of the excipient and particular needs of thecomposition. Typically, the optimal amount of any individual excipientis determined through routine experimentation, i.e., by preparingcompositions containing varying amounts of the excipient (ranging fromlow to high), examining stability and other parameters of thecomposition, and then determining the range at which optimal performanceis attained with no significant adverse effects.

Generally, however, the excipient will be present in the composition inan amount of about 1% to about 99% by weight, preferably from about5%-98% by weight, more preferably from about 15-95% by weight of theexcipient, with concentrations less than 30% by weight most preferred.

These foregoing pharmaceutical excipients along with other excipientsare described in “Remington: The Science & Practice of Pharmacy”,19^(th) ed., Williams & Williams, (1995), the “Physician's DeskReference”, 52^(nd) ed., Medical Economics, Montvale, N.J. (1998), andKibbe, A. H., Handbook of Pharmaceutical Excipients, 3^(rd) Edition,American Pharmaceutical Association, Washington, D.C., 2000.

The pharmaceutical preparations of the present invention are typically,although not necessarily, administered via injection and are thereforegenerally liquid solutions or suspensions immediately prior toadministration. The pharmaceutical preparation can also take other formssuch as syrups, creams, ointments, tablets, powders, and the like. Othermodes of administration are also included, such as pulmonary, rectal,transdermal, transmucosal, oral, intrathecal, subcutaneous,intra-arterial, and so forth.

As previously described, the conjugates can be administered parenterallyby intravenous injection, or less preferably by intramuscular or bysubcutaneous injection. Suitable formulation types for parenteraladministration include ready-for-injection solutions, dry powders forcombination with a solvent prior to use, suspensions ready forinjection, dry insoluble compositions for combination with a vehicleprior to use, and emulsions and liquid concentrates for dilution priorto administration, among others.

The invention also provides a method for administering a conjugate asprovided herein to a patient suffering from a condition that isresponsive to treatment with a conjugate. The method comprisesadministering, generally via injection, a therapeutically effectiveamount of the conjugate (preferably provided as part of a pharmaceuticalpreparation). The method of administering may be used to treat anycondition that can be remedied or prevented by administration of theparticular conjugate. Those of ordinary skill in the art appreciatewhich conditions a specific conjugate can effectively treat. The actualdose to be administered will vary depend upon the age, weight, andgeneral condition of the patient as well as the severity of thecondition being treated, the judgment of the health care professional,and conjugate being administered. Therapeutically effective amounts areknown to those skilled in the art and/or are described in the pertinentreference texts and literature. Generally, a therapeutically effectiveamount will range from about 0.001 mg to 100 mg, preferably in dosesfrom 0.01 mg/day to 75 mg/day, and more preferably in doses from 0.10mg/day to 50 mg/day.

The unit dosage of any given conjugate (again, preferably provided aspart of a pharmaceutical preparation) can be administered in a varietyof dosing schedules depending on the judgment of the clinician, needs ofthe patient, and so forth. The specific dosing schedule will be known bythose of ordinary skill in the art or can be determined experimentallyusing routine methods. Exemplary dosing schedules include, withoutlimitation, administration five times a day, four times a day, threetimes a day, twice daily, once daily, three times weekly, twice weekly,once weekly, twice monthly, once monthly, and any combination thereof.Once the clinical endpoint has been achieved, dosing of the compositionis halted.

It is to be understood that while the invention has been described inconjunction with the preferred specific embodiments thereof, that theforegoing description as well as the examples that follow are intendedto illustrate and not limit the scope of the invention. Other aspects,advantages and modifications within the scope of the invention will beapparent to those skilled in the art to which the invention pertains.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

All articles, books, patents and other publications referenced hereinare hereby incorporated by reference in their entireties.

Experimental

The practice of the invention will employ, unless otherwise indicated,conventional techniques of organic synthesis, biochemistry, proteinpurification and the like, which are within the skill of the art. Suchtechniques are fully explained in the literature. See, for example, J.March, Advanced Organic Chemistry: Reactions Mechanisms and Structure,4th Ed. (New York: Wiley-Interscience, 1992), supra.

In the following examples, efforts have been made to ensure accuracywith respect to numbers used (e.g., amounts, temperatures, etc.) butsome experimental error and deviation should be accounted for. Unlessindicated otherwise, temperature is in degrees C and pressure is at ornear atmospheric pressure at sea level. Each of the following examplesis considered to be instructive to one of ordinary skill in the art forcarrying out one or more of the embodiments described herein. All ¹H NMRdata was generated by a 300 or 400 MHz NMR spectrometer manufactured byBruker. In Examples 5 through 12, commercial grade mPEG(20 k Da)-aminewas used having the following characterization: percent substitution ofamine, 94.6 to 100%; percent hydroxy mPEG impurity, 0 to 4.2%; percentdimer (species formed from the reaction of two functionalized PEGspecies to each other), 0.6 to 2.1%; percent trimer (species formed fromthe reaction of three functionalized PEG species to each other), 0 to0.3%.

EXAMPLE 1 Preparation of a Substituted Maleamic Acid-Terminated,Water-Soluble Polymer

To a solution of mPEG (20 k Da)-amine (Nektar Therapeutics, 50.0 g,0.0025 mol) in anhydrous dichloromethane (350 ml),N-methoxycarbonylmaleimide (0.80 g, 0.0051 mol) was added and thesolution was stirred for one hour at room temperature under argonatmosphere. N,N-diisopropylethylamine (1.0 ml) was added and the mixturewas stirred overnight at room temperature under argon atmosphere. Nextthe reaction mixture was concentrated by distilling off ˜200 mldischloromethane and the product was precipitated with ethyl ether.Yield after drying 46.3 g. NMR (d₆-DMSO): 3.24 ppm (s, PEG—OCH₃), 3.51ppm (s, PEG backbone), 3.86 ppm (s,CH₃O—NH—), 6.20 ppm (m, —CH═CH—),8.46 ppm (—NH).

EXAMPLE 2 Preparation of mPEG (20 k Da)Maleimide

To the solution of the substituted maleamic acid-terminated,water-soluble polymer prepared in Example 1 (10.0 g), in anhydrousacetonitrile (100 ml) N,N-diisopropylethylamine (10 ml) was added andthe reaction mixture was stirred for forty-four hours at roomtemperature under argon atmosphere. Next, the mixture was concentratedby distilling off ˜80 ml acetonitrile and the product was precipitatedwith ethyl ether giving 8.5 g of mPEG_((20K Da))maleimide. NMR(d₆-DMSO): 3.24 ppm (s, PEG—OCH₃), 3.51 ppm (s, PEG backbone), 7.01 ppm(s, —CH═CH—, maleimide); substitution 93.5%.

EXAMPLE 3 A Protocol for Preparing Substituted Maleamic Acid-Terminated,Water-Soluble Polymer

wherein (with respect to Formula II):

POLY is a water-soluble polymer (preferably linear or branched, andpreferably is CH₃O—(CH₂CH₂O)_(n)—CH₂CH₂—, wherein (n) is 2 to 4000 whenPOLY is linear);

(c) is zero or one (preferably zero); and

X², wherein present, is a spacer moiety,

wherein (with respect to Formula III):

Y¹ is O or S;

Y² is O or S;

(a) is an integer from 1 to 20;

R¹, in each instance, is independently H or an organic radical;

R², in each instance, is independently H or an organic radical;

R³, in each instance, is independently H or an organic radical; and

R⁴, in each instance, is independently H or an organic radical,

wherein (with respect to Formula I):

POLY is a water-soluble polymer;

(b) is zero or one;

X¹, wherein present, is a spacer moiety;

Y¹ is O or S;

Y² is O or S;

(a) is an integer from 1 to 20;

R¹, in each instance, is independently H or an organic radical;

R², in each instance, is independently H or an organic radical;

R³, in each instance, is independently H or an organic radical; and

R⁴, in each instance, is independently H or an organic radical.

Dissolve the amine-terminated, water-soluble polymer (Formula II) intodichloromethane (20% wt/v solution) and distill under reduced pressureat 40° C. until all the dichloromethane is removed. This will form anazeotropic mixture with water and effectively remove the water from theremaining polymer. Repeat this step once more. Place under vacuum to drycompletely to a solid if desired (not necessary).

Redissolve the polymer in anhydrous dichloromethane (20% wt/v solution)under an inert gas atmosphere. Add 1.5 equivalents of a maleimidereagent (Formula III). Once dissolved, add 0.5 equivalents ofdiisopropylethylamine dropwise. Let stir at room temperature under inertatmosphere for at least one hour (overnight is fine, but may formclosed-ring maleimide).

Distill off solvent under reduced pressure at 25-30° C. until a thickoil-like solution results (approximately 0.5-1.5 mL of solution per gramof water-soluble polymer, depending upon molecular weight). Addisopropyl alcohol slowly to the stirring solution (approximately 25 mL/gof water-soluble polymer). Let stir at room temperature for at leastthirty minutes. Filter off the liquid. Add back enough isopropyl alcoholto make a slurry of the water-soluble polymer, and then filter off theliquid once again. Dry the solids under vacuum until all isopropylalcohol is removed.

EXAMPLE 4 A Protocol For Preparing a Maleimide-Terminated, Water-SolublePolymer

wherein (with respect to Formula I):

POLY is a water-soluble polymer;

(b) is zero or one;

X¹, wherein present, is a spacer moiety;

Y¹ is O or S;

Y² is O or S;

(a) is an integer from 1 to 20;

R¹, in each instance, is independently H or an organic radical;

R², in each instance, is independently H or an organic radical;

R³, in each instance, is independently H or an organic radical; and

R⁴, in each instance, is independently H or an organic radical,

wherein (with respect to Formula V) each of POLY, X² and (c) are definedas provided in Formula II and each of R³ and R⁴ are defined as providedin Formula III.

Dissolve the substituted maleamic acid-terminated, water-soluble polymerfrom Example 3 into anhydrous dichloromethane to make a 10% wt/vsolution. Add anhydrous sodium sulfate (0.5 g/g of PEG). Add anhydroussodium carbonate (0.5 g/g of water-soluble polymer). Heat to refluxunder inert gas atmosphere (approximately 40 ° C.). Stir at reflux forfive hours. Remove heat and let cool to less than 35° C. Filter offsolids. Distill off solvent under reduced pressure at 25-40° C. until athick oil-like solution results. Precipitate with isopropyl alcohol asin Example 3.

EXAMPLE 5 Nonaqueous Preparation of a Maleimide-Terminated,Water-Soluble Polymer

Azeotroped mPEG(20 k Da)-amine, 0.01 wt % butylated hydroxytoluene(BHT), and dichloromethane were combined at 40° C. Evaporation of themore volatile components was conducted using a rotary evaporator. Thewater content was tested and found to be 56 ppm (below 100 ppm isdesired). To this mixture was added 0.5 g/g each of milled sodiumcarbonate and granular sodium sulfate. Following addition of the milledsodium carbonate and granular sodium sulfate, the mixture was stirredand cooled to 5° C. to form a cold PEG solution.

Separately, 3 eq (0.56 g) of N-methoxycarbonylmaleimide was dissolved indichloromethane to make a 3% (w/v) solution. The resulting mixture wasvortexed for 30 seconds. The vortexed mixture had a cloudy appearance.The vortexed mixture was added to the cold PEG solution and stirred for21 hours at or about 5° C.

Following stirring, the mixture was heated gradually to about 40 ° C.and refluxed for over 45 minutes. Thereafter, samples were withdrawn todetermine reaction completion by H NMR

After 8.5 hours at reflux, the mixture was cooled to room temperature,filtered through a celite bed, and followed by removal of thedichloromethane solvent using a rotary evaporator and a bath at 30° C.,thereby producing the an oil. The product was recovered by precipitationwith isopropyl alcohol (IPA) (stirring for 30 minutes).

Analyses associated with this example are provided in Tables 1 and 2 anddiscussed in Example 13.

EXAMPLE 6 Nonaqueous Preparation of a Maleimide-Terminated,Water-Soluble Polymer

The procedure of Example 5 was repeated, with the followingexceptions/notations. Following addition of the milled sodium carbonateand granular sodium sulfate, the mixture was cooled to 5° C. for 10hours. Reflux was conducted for 7 hours (which showed 6% precursor atthe 6^(th) hour).

Analyses associated with this example are provided in Tables 1 and 2 anddiscussed in Example 13.

EXAMPLE 7 Nonaqueous Preparation of a Maleimide-Terminated,Water-Soluble Polymer

The procedure of Example 5 was repeated, with the followingexceptions/notations. Following addition of the milled sodium carbonateand granular sodium sulfate, the mixture was stirred and cooled to 5.7°C. for 9.5 hours. Rather than heating and then refluxing, the mixturewas stirred at room temperature for 45 minutes prior to refluxing.Reflux was conducted for 6.5 hours (which showed 6% precursor at the 5thhour).

Analyses associated with this example are provided in Tables 1 and 2 anddiscussed in Example 13.

EXAMPLE 8 Nonaqueous Preparation of a Maleimide-Terminated,Water-Soluble Polymer

The procedure of Example 5 was repeated, with the followingexceptions/notations. Following addition of the milled sodium carbonateand granular sodium sulfate, the mixture was stirred and cooled to 5.75°C. initially and gradually cooled over 2 hours to 5° C. with stirringfor 15 hours total. Rather than heating and then refluxing, the mixturewas stirred at room temperature for 1 hour prior to refluxing. Refluxwas conducted for 8 hours (which showed 12% precursor at the 6^(th)hour).

Analyses associated with this example are provided in Tables 1 and 2 anddiscussed in Example 13.

EXAMPLE 9 (COMPARATIVE EXAMPLE) “Aqueous N-AlkoxycarbonylmaleimideRoute”

A 17.5% solution (w/v) of mPEG amine (20 k Da) in 7.6% (w/v) sodiumbicarbonate solution was cooled to 6° C. A 10% solution (w/v) ofN-methoxycarbonylmaleimide (3 eq., 5.3%) in acetonitrile was added andthe mixture and stirred for 15 minutes. Enough distilled water was addedto the solution to double the volume. The solution was first cooled andthen allowed to warm to 13° C. over 45 minutes.

The pH of the solution was adjusted to 3.0 with phosphoric acid and thenenough sodium chloride was added to provide a salt solution of 15%sodium chloride (w/v). The salt solution was stirred for 15 minutes andthen extracted with an equivalent volume of dichloromethane, therebyproviding a dichloromethane solution.

The dichloromethane solution was dried with sodium sulfate (3.5 g /100mL) and evaporated to result in an oil. Precipitation with isopropylalcohol (17.5 mL/g), filtration and drying gave a white solid.

Analyses associated with this example are provided in Tables 1 and 2 anddiscussed in Example 13.

EXAMPLE 10 (Comparative Example) “Aqueous N-AlkoxycarbonylmaleimideRoute”

A 17.5% solution (w/v) of mPEG amine (20 k Da) in 7.6% (w/v) sodiumbicarbonate solution was cooled to 3.8 to 5.9° C. An excursion intemperature to room temperature overnight occurred due to chillerproblems. A 10% solution (w/v) of N-methoxycarbonylmaleimide (3 eq.,4.4%) in acetonitrile was added and the mixture and stirred for 15minutes. Enough distilled water was added to the solution to double thevolume. The solution was first cooled and then allowed to warm to 13° C.over 45 minutes.

The pH of the solution was adjusted to 3.0 with phosphoric acid and thenenough sodium chloride was added to provide a salt solution of 15%sodium chloride (w/v). The salt solution was stirred for 15 minutes andthen extracted with an equivalent volume of dichloromethane, therebyproviding a dichloromethane solution.

The dichloromethane solution was dried with sodium sulfate (3.5 g /100mL) and evaporated to result in an oil. Precipitation with isopropylalcohol (17.5 mL/g), filtration and drying gave a white solid.

Analyses associated with this example are provided in Tables 1 and 2 anddiscussed in Example 13.

EXAMPLE 11 (Comparative Example) “Aqueous N-AlkoxycarbonylmaleimideRoute”

A 17.5% solution (w/v) of MPEG amine (20 k Da) in 7.6% (w/v) sodiumbicarbonate solution was cooled to 4° C. A 10% solution (w/v) ofN-methoxycarbonylmaleimide (3 eq., 5.4%) in acetonitrile was added andthe mixture and stirred for 15 minutes. Enough distilled water was addedto the solution to double the volume. The solution was first cooled andthen allowed to warm to 8 to 9° C. over 45 minutes.

The pH of the solution was adjusted to 3.0 with phosphoric acid and thenenough sodium chloride was added to provide a salt solution of 15%sodium chloride (w/v). The salt solution was stirred for 15 minutes andthen extracted with an equivalent volume of dichloromethane, therebyproviding a dichloromethane solution.

The dichloromethane solution was dried with sodium sulfate (3.5 g /100mL) and evaporated to result in an oil. Precipitation with isopropylalcohol (17.5 mL/g), filtration and drying gave a white solid.

Analyses associated with this example are provided in Tables 1 and 2 anddiscussed in Example 13.

EXAMPLE 12 (Comparative Example) “Aqueous N-AlkoxycarbonylmaleimideRoute”

A 17.5% solution (w/v) of MPEG amine (20 k Da) in 7.6% (w/v) sodiumbicarbonate solution was cooled to 6° C. A 10% solution (w/v) ofN-methoxycarbonylmaleimide (3 eq., 5.3%) in acetonitrile was added andthe mixture and stirred for 15 minutes. Enough distilled water was addedto the solution to double the volume. The solution was first cooled andthen allowed to warm to 13° C. over 45 minutes.

The pH of the solution was adjusted to 3.0 with phosphoric acid and thenenough sodium chloride was added to provide a salt solution of 15%sodium chloride (w/v). The salt solution was stirred for 15 minutes andthen extracted with an equivalent volume of dichloromethane, therebyproviding a dichloromethane solution.

The dichloromethane solution was dried with sodium sulfate (3.5 g /100mL) and evaporated to result in an oil. Precipitation with isopropylalcohol (17.5 mL/g), filtration and drying gave a white solid.

Analyses associated with this example are provided in Tables 1 and 2 anddiscussed in Example 13.

EXAMPLE 13

The products obtained from Examples 5 to 12 were analyzed using HPLC,GFC and ¹H NMR. High Performance Liquid Chromatography (HPLC) wasperformed using an Agilent 1100 HPLC system (Agilent) using a ShodexProtein KW-803 GFC column with a mobile phase of 10 mM HEPES, flow rateof 1.0 mL/minute and temperature of 25° C. with use of an RI detector(product derivatized with a carboxylic acid functionalized thiolspecies, and substitution is determined by comparison of derivatized andunderivatized spectra). GFC was performed using a Shodex Protein KW-803GFC column with a mobile phase of 1× phosphate-buffered saline, flowrate of 1.0 mL/minute and temperature of 25° C. with use of an RIdetector. The results are provided in Tables 1 and 2. With regard toTable 1, “% of Dimaleimidyl species” refers to a polymer of about thesame molecular weight as the desired maleimide product, but with twomaleimidyl termini, and with regard to Table 2, “% Dimer of MAL” refersto species formed from the reaction of two functionalized PEG species toeach other, % Trimer of MAL refers to species formed from the reactionof three functionalized PEG species to each other, and “% Higher MW thanTimer” refers to species formed from the reaction of four or morefunctionalized PEG species to each other. TABLE 1 HPLC Analyses of theProducts From Examples 5 to 12 HPLC % of % of Unknowns + % mPEG- mPEG- %of % of Product from Substitution Maleamic Maleamic UnreactedDimaleimidyl Example # of Maleimide Acid Acid species species Example 589 ≦1.9 6.8* 4 3.7 Example 6 86 ≦1.1 6.6* 7.5 2.6 Example 7 89 ≦1.9 4.6*6 2.5 Example 8 88 ≦1.4 3.1* 6.3 2.5 Example 9 83 7.9 9.7* 5.9 3.8(Comparative) Example 10 *** 0 65 — — — (Comparative) Example 11 86 4.54.5* 7.7 1.8 (Comparative) Example 12 80 13.6 14.2*  4 2 (Comparative)*Includes the mPEG-maleamic peak, but not unreacted species*** Example experienced a temperature excursion

TABLE 2 GFC and ¹H NMR Analyses of the Products From Examples 5 to 12and Yields and Batch Sizes of the Products From Examples 5 to 8 GFC (GelFiltration Chromatography) ¹H NMR % % % % of substituted % Open- BatchProduct from Dimer of Trimer Higher MW maleamic acid ring ester MassSize Example # MAL of MAL than trimer species impurity Yield (%) (g)Example 5 3.2 1.3 1.1 0 0 85 ˜25 Example 6 4.5 1.2 0 2 2.3 88 ˜25Example 7 2.8 0.4 0.3 ≦1.5** ≦2.0** 79 ˜25 Example 8 2.3 0.4 0 0 0 85˜25 Example 9 1.6 0 0 0 0 93.5 ˜1122 (Comparative) Example 10*** — — — —— 66.4 ˜797 (Comparative) Example 11 0.9 0 0.6 5.8 0 90.7 ˜463(Comparative) Example 12 0.7 0 0 0 0 87 ˜1045 (Comparative)**Not quantifiable, but value provided is the expected value***Example experienced a temperature excursion

From Table 1, it is clear that the general method employed in Examples 5though 8 resulted in compositions having greater maleimide substitutionthan the composition generated in accordance with the aqueous-basedapproach followed in comparative Examples 9 through 12. Often, thegeneral method employed in Examples 5 through 8 provided maleimidesubstitution greater than 86 percent. Also, Table 1 demonstrates thegeneral method employed in Examples 5 though 8 resulted in compositionshaving a percentage of polymers bearing a maleamic acid (“mPEG-maleamicacid”) of less than 4 percent, and even less than 2 percent, which isbetter than could be achieved with the aqueous-based approach followedin comparative Examples 9 through 12.

From Table 2, it appears that with the exception higher molecular weightspecies analyzed through gas filtration chromatography (% dimer ofmaleimide, % trimer of maleimide and other high molecular weightspecies), parameters such as the percentages of M-MAL 20 k precursor andopen-ring ester impurity are fairly consistent among the examplestested.

Finally, it is clear from Tables 1 and 2 that the general methodemployed in Examples 5 though 8 provided consistent and good yields whencompared to general method employed in the comparative Examples.

1. A synthetic method comprising: a) combining an amine-terminated,water-soluble polymer with a maleimide reagent under substantiallynonaqueous conditions to form a substituted maleamic acid-terminated,water-soluble polymer; and b) exposing the maleamic acid-terminated,water-soluble polymer to elimination conditions to thereby result in amaleimide-terminated, water-soluble polymer.
 2. The method of claim 1,further comprising isolating the substituted maleamic acid-terminated,water-soluble polymer is prior to exposing the maleamic acid-terminated,water-soluble polymer to elimination conditions.
 3. The method of claim2, wherein isolating the maleamic acid-terminated, water-soluble polymeris effected by precipitation to prepare a dried maleamicacid-terminated, water-soluble polymer.
 4. The method of claim 3,wherein precipitation is effected by adding an excess of an agentselected from the group consisting of isopropyl alcohol, diethyl ether,MTBE, heptane, THF, hexane, and mixtures thereof.
 5. The method of claim3, further comprising the step of dissolving the dried maleamicacid-terminated, water-soluble polymer to regenerate the maleamicacid-terminated, water-soluble polymer.
 6. The method of claim 1,carried out in an organic solvent.
 7. The method of claim 6, wherein theorganic solvent is selected from the group consisting of halogenatedaliphatic hydrocarbons, alcohols, aromatic hydrocarbons, halogenatedaliphatic hydrocarbons, alcohols, aromatic hydrocarbons, halogenatedaromatic hydrocarbons, amides, nitrites, ketones, acetates, ethers,cyclic ethers, and combinations thereof.
 8. The method of claim 6,wherein the organic solvent is selected from the group consisting ofdichloromethane, chloroform, acetonitrile, toluene, methyl t-butylether, tetrahydrofuran, octanol, ethyl acetate, diethylcarbonate,acetone, cyclohexane and combinations thereof.
 9. The method of claim 8,wherein the organic solvent is dichloromethane or acetonitrile.
 10. Themethod of claim 1, wherein the step of combining an amine-terminated,water-soluble polymer with a maleimide reagent under substantiallynonaqueous conditions to form maleamic acid-terminated, water-solublepolymer is carried out in the presence of basic catalyst.
 11. The methodof claim 1, wherein the step of exposing the maleamic acid-terminated,water-soluble polymer to elimination conditions in the organic solventin the presence of a base.
 12. The method of claim 11, wherein the baseis selected from the group consisting of sodium bicarbonate, potassiumbicarbonate, sodium carbonate, or potassium carbonate.
 13. The method ofclaim 11, wherein the step of exposing the maleamic acid-terminated,water-soluble polymer to elimination conditions at a temperature 10 to60° C.
 14. The method of claim 1, wherein the step of exposing themaleamic acid-terminated, water-soluble polymer to eliminationconditions is carried out in the presence of a non-nucleophilic aminecatalyst.
 15. The method of claim 14, wherein the non-nucleophilic aminecatalyst is selected from the group consisting of diisopropylethylamine,triethylamine, n-methyl morpholine, pyridine,N,N-Dimethyl-4-aminopyridine, 1,8-Diazabicyclo[5.4.0]undec-7-ene, and1,4-diazabicyclo[2.2.2]octane.
 16. The method of claim 1, wherein thestep of exposing the maleamic acid-terminated, water-soluble polymer toelimination conditions comprises is carried out in the presence of adrying agent.
 17. The method of claim 16, wherein the drying agent isselected from the group consisting of NaHCO₃, Na₂CO₃, CaCl₂, CaSO₄,MgSO₄, KOH, Na₂SO₄, K₂CO₃, KHCO₃, molecular sieves and combinationsthereof.
 18. The method of claim 2, wherein the amine-terminated,water-soluble polymer is a water-soluble polymer bearing a terminalamine group, wherein the water-soluble polymer is selected from thegroup consisting of poly(alkylene glycol), poly(olefinic alcohol),poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide),poly(hydroxyalkylmethacrylate), poly(saccharide), poly(α-hydroxyaceticacid), poly(acrylic acid), poly(vinyl alcohol), polyphosphazene,polyoxazoline, poly(N-acryloylmorpholine), and copolymers or terpolymersthereof,
 19. The method of claim 18, wherein the water-soluble polymeris a poly(ethylene glycol).
 20. The method of claim 19, wherein thepoly(ethylene glycol) has a molecular weight of about 100 to about100,000 Daltons.
 21. The method of claim 1, wherein the maleimidereagent is of the formula:

wherein: Y¹ is O or S; Y² is O or S; (a) is an integer from 1 to 20; R¹,in each instance, is independently H or an organic radical; R², in eachinstance, is independently H or an organic radical; R³, in eachinstance, is independently H or an organic radical; and R⁴, in eachinstance, is independently H or an organic radical.
 22. The method ofclaim 1, wherein the maleamic acid-terminated, water-soluble polymer isof the formula:

wherein: POLY is a water-soluble polymer; (b) is zero or one; X¹,wherein present, is a spacer moiety; Y¹ is O or S; Y² is O or S; (a) isan integer from 1 to 20; R¹, in each instance, is independently H or anorganic radical; R², in each instance, is independently H or an organicradical; R³, in each instance, is independently H or an organic radical;and R⁴, in each instance, is independently H or an organic radical. 23.The method of claim 22, wherein POLY is a linear water-soluble polymer.24. The method of claim 22, wherein POLY is CH₃O—(CH₂CH₂O)_(n)—CH₂CH₂and (a) is one, and further wherein (n) is 2 to
 4000. 25. The method ofclaim 22, wherein POLY is branched.
 26. The method of claim 1, whereinthe maleimide-terminated, water-soluble polymer is of the formula:

wherein: POLY is a water-soluble polymer; (b) is zero or one; X¹,wherein present, is a spacer moiety; R³, in each instance, isindependently H or an organic radical; and R⁴, in each instance, isindependently H or an organic radical.
 27. The method of claim 26,wherein POLY is a linear water-soluble polymer.
 28. The method of claim26, wherein POLY is CH₃O—(CH₂CH₂O)_(n)—CH₂CH₂—, wherein (n) is 2 to4000.
 29. The method of claim 26, wherein POLY is branched.
 30. Themethod of claim 1, wherein the substantially nonaqueous conditionsrepresents a reaction medium having less 1000 parts per million ofwater.
 31. The method of claim 30, wherein the substantially nonaqueousconditions represents a reaction medium having less than 100 parts permillion of water.
 32. The method of claim 31, wherein the substantiallynonaqueous conditions represents a reaction medium having less than 60parts per million of water.
 33. The method of claim 1, wherein acomposition is formed in which greater than 70 percent of thewater-soluble polymer species in the composition aremaleimide-terminated, water-soluble polymers.
 34. The method of claim 1,wherein a composition is formed in which greater than 80 percent of thewater-soluble polymer species in the composition aremaleimide-terminated, water-soluble polymers.
 35. The method of claim 1,wherein a composition is formed in which greater than 90 percent of thewater-soluble polymer species in the composition aremaleimide-terminated, water-soluble polymers.
 36. The method of claim 1,wherein a composition is formed in which greater than 93 percent of thewater-soluble polymer species in the composition aremaleimide-terminated, water-soluble polymers.
 37. The method of claim 1,wherein a composition is formed in which greater than 86 percent of thewater-soluble polymer species in the composition are mPEG-maleimides.38. The method of claim 1, wherein a composition is formed in whichgreater than 93 percent of the water-soluble polymer species in thecomposition are mPEG-maleimides.
 39. The composition resulting fromcarrying out the method of claim
 1. 40. A method for preparing aconjugate, comprising: combining, under conjugation conditions, athiol-containing biologically active agent with a maleimide-terminated,water-soluble polymer prepared by (i) combining an amine-terminated,water-soluble polymer with a maleimide reagent under substantiallynonaqueous conditions to form a substituted maleamic acid-terminated,water-soluble polymer, and (ii) exposing the maleamic acid-terminated,water-soluble polymer to elimination conditions to thereby result in amaleimide-terminated, water-soluble polymer.
 41. The conjugate preparedaccording to claim
 40. 42. A composition comprising polymeric specieswherein at least 70% of the polymeric species in the composition aremaleimide-terminated, water-soluble polymers and further wherein thecomposition comprises open ring ester polymeric species.